Norbornane type ester hydrolase from Acetobacter pasteurianus

ABSTRACT

A norbornane type ester hydrolase that enantio-selectively hydrolyzes a (±)-exo-norbornane type ester represented by Formula I is provided: ##STR1## wherein R is acyl, and A and B are hydrogens, respectively, or where A and B are absent, resulting in a carbon-carbon double bond between the carbons to which A and B are attached in Formula I. The norbornane type ester hydrolase has an optimal pH of approximately 8 and a stable pH range of approximately 6 to 8.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel norbornane type ester hydrolasethat enantio-selectively hydrolyzes a (±)-exo-norbornane type ester toproduce optically active norborneol; a structural gene of the norbornanetype ester hydrolase; an expression vector including the structuralgene; a transformant including the expression vector; and a productionmethod for the norbornane type ester hydrolase using the transformant.

2. Description of the Related Art

Several methods for preparing norborneol are known in the art. The knownmethods are classified into chemical and biological methods. Forexample, the following chemical method is known. Norbornene used as astarting material is allowed to react with an organic acid to produce anorbornane type ester compound. The ester compound is chemicallyhydrolyzed to produce norborneol. In this chemical method, four kinds ofstereoisomers of norborneol (i.e., (+)-endo-norborneol,(-)-endo-norborneol, (+)-exo-norborneol and (-)-exo-norborneol) aresimultaneously produced. Therefore, a complicated separation is furtherrequired to obtain an optically active norborneol.

Another production method for norborneol using the following biologicalmethod is also known. Norbornene used as a starting material is allowedto react with an organic acid to produce a norbornane type estercompound. The ester compound is allowed to react with an enzyme, or tocome in contact with microorganisms producing such an enzyme, therebyhydrolyzing the ester compound to obtain norborneol. With regard to thisbiological method, Oberhauser et al. reported a method for producing(-)-norborneol from (±)-norbornyl acetate by using a lipase derived fromCandida cylindraceae (Th. Oberhauser et al., Tetrahedron, 43, 3931-3941,1987). The method using the lipase, however, has a low selectivity andthe produced norborneol has a low optical purity.

Japanese Laid-Open Patent Publication No. 2-273196 discloses an opticalresolution method of a racemic mixture using a biological hydrolysisreaction, in which an inhibitor for selectively inhibiting thehydrolysis reaction of one of the enantiomers is used in the reaction.Such a method can be employed to prepare optically active norborneol. Inthis method, however, it is necessary to perform a screening ofinhibitors to obtain an inhibitor that is useful in the selection of theoptically active norborneol. Further, it is required to remove theinhibitor after the reaction. As a result, such a method isdisadvantageously complicated.

SUMMARY OF THE INVENTION

The norbornane type ester hydrolase of this inventionenantio-selectively hydrolyzes a (±)-exo-norbornane type esterrepresented by Formula I: ##STR2##

wherein R is acyl, and A and B are hydrogens, respectively, or where Aand B are absent, resulting in a carbon-carbon double bond between thecarbons to which A and B are attached in Formula I;

the norbornane type ester hydrolase having an optimal pH ofapproximately 8 and a stable pH range of approximately 6 to 8.

A norbornane type ester hydrolase of this invention enantio-selectivelydeacetylates a (±)-exo-norbornyl acetate represented by Formula II:##STR3##

the norbornane type ester hydrolase having an optimal pH ofapproximately 8 and a stable pH range of approximately 6 to 8.

In one embodiment, a norbornane type ester hydrolase of this inventionis derived from a bacterium of the genus Acetobacter, preferably,Acetobacter pasteurianus.

In one embodiment, the above-mentioned Acetobacter pasteurianus isAcetobacter pasteurianus ATCC 12873.

A norbornane type ester hydrolase of this invention comprises an aminoacid sequence from Met in the 1 position to Ala in the 388 position ofSEQ ID No. 1.

The present invention includes a DNA sequence encoding the norbornanetype ester hydrolase having the above-mentioned amino acid sequence.

In one embodiment, the above-mentioned DNA sequence comprises a basesequence from A in the 1 position to C in the 1164 position of SEQ IDNo. 1.

The present invention includes an expression vector having theabove-mentioned DNA sequence.

The present invention includes a transformant produced by introducingthe above-mentioned expression vector into a host.

In one embodiment, the host is E. coli.

A production method for a norbornane type ester hydrolase of thisinvention comprises the steps of, culturing the above-mentionedtransformant in a culture medium, and recovering the produced norbornanetype ester hydrolase from the culture.

A production method for an optically active norborneol of this inventioncomprises the step of, allowing a (±)-exo-norbornane type ester to comein contact with the above-mentioned norbornane type ester hydrolase; the(±)-exo-norbornane type ester being represented by Formula I: ##STR4##wherein R is acyl, and A and B are hydrogens, respectively, or where Aand B are absent, resulting in a carbon-carbon double bond between thecarbons to which A and B are attached in Formula I.

In one embodiment, R in Formula I is acetyl, and A and B are hydrogens,respectively.

Thus, the invention described herein makes possible the advantages of(1) providing a novel norbornane type ester hydrolase that can produceoptically active norborneol with a high purity; and (2) providing a DNAsequence for encoding the norbornane type ester hydrolase, an expressionvector including the DNA sequence, a transformant including theexpression vector and a production method for the norbornane type esterhydrolase using the transformant.

These and other advantages of the present invention will become apparentto those skilled in the art upon reading and understanding the followingdetailed description with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows enantio-selective deacetylation of (±)-exo-norbornylacetate by a norbornane type ester hydrolase of the present invention.

FIG. 2 is a restriction map of a plasmid pBNA21 including the structuralgene of the norbornane type ester hydrolase of the present invention.

FIG. 3 is a restriction map of a plasmid pUNA221 including thestructural gene of the norbornane type ester hydrolase of the presentinvention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventors have conducted various studies to obtain opticallyactive norborneol with a high purity from a racemic mixture ofnorbornane type ester compounds. As a result, it was found that a strainof Acetobacter pasteurianus ATCC 12873 produces a norbornane type esterhydrolase, and that the hydrolase selectively deacetylates(±)-exo-norbornyl acetate as shown in FIG. 1 to produce optically activenorborneol with a high optical purity. The present inventors havefurther determined a DNA sequence for encoding the norbornane type esterhydrolase in the genomic DNA of the strain, thereby achieving thepresent invention.

The term "optically active norborneol" as used herein means one of thecompounds (+)-norborneol and (-)-norborneol. A preferable opticallyactive norborneol is (-)-norborneol.

The term "a norbornane type ester" as used herein means an ester ofnorborneol or an ester of norborneol derivatives. The (±)-exo-norbornanetype ester used in the present invention is represented by Formula I:##STR5## wherein R is acyl; and A and B are hydrogens, respectively, orwhere A and B are absent, resulting in a carbon-carbon double bondbetween the carbons to which A and B are attached in Formula I.

In Formula I, the acyl is an aliphatic acyl, cycloalkylcarbonyl orarylcarbonyl. The aliphatic acyl has 2 to 10 carbon atoms, andpreferably 2 to 7 carbon atoms. The aliphatic acyl includes formyl,acetyl, propionyl, butyryl, isobutyryl, pentanoyl and hexanoyl, and ismore preferably formyl, acetyl, propionyl or isobutyryl. Thecycloalkylcarbonyl preferably has 4 to 10 carbon atoms, and preferably 4to 7 carbon atoms. The cycloalkylcarbonyl includes cyclopropanecarbonyl,cyclobutanecarbonyl, cyclopentanecarbonyl and cyclohexanecarbonyl. Thearylcarbonyl preferably has 7 to 11 carbon atoms and includes benzoyl,p-toluoyl and naphtoyl.

The compounds of Formula I are commercially available or alternativelycan be easily chemically synthesized with methods known in the art. Forexample, a desired (±)-exo-norbornane type ester represented by FormulaI can be produced in a high yield by reacting norbornene with anappropriate organic acid such as formic acid, acetic acid, propionicacid and lactic acid in the presence of an acid catalyst; or by causinga Dieis-Alder reaction between a vinyl ester of an appropriate organicacid and cyclopentadiene.

The norbornane type ester hydrolase of the present invention is producedfrom bacteria of genus Acetobacter, preferably Acetobacter pasteurianusATCC 12873. This strain is available from American Type CultureCollection (ATCC).

(1) Culture conditions:

No special medium is required for the cultivation of the above-mentionedbacteria, and any of the various conventional types of culture mediumscan be used. For example, a medium containing glucose, peptone, yeastextract, various salts and the like can be used. The appropriate mediumpH is 5 to 9, and preferably approximately 7. The appropriate mediumtemperature is 25° to 30° C., and preferably approximately 28° C. Thebacteria are cultured, for example, aerobically with stirring orshaking. The norbornane type ester hydrolase of the present invention isproduced intracellularly.

(2) Purification of the enzyme:

One or a combination of known techniques can be used to isolate andpurify the norbornane type ester hydrolase from the culture. Forexample, the culture is centrifuged to collect bacteria cells therein.The cells of the collected bacteria are disrupted, and the resultant iscentrifuged again to obtain a supernatant as a crude enzyme solution.The crude enzyme solution is purified by an appropriate method to obtainthe norbornane type ester hydrolase. For example, the crude enzymesolution is subjected to a DEAE Sepharose chromatography and then topolyacrylamide gel electrophoresis to isolate a band exhibiting anenzymatic activity, thus, obtaining the norbornane type ester hydrolase.

(3) Measurement of the enzymatic activity:

The norbornane type ester hydrolase reacts with p-nitrophenyl acetate,in addition to exo-norbornane type ester, as a substrate to produce acolored material, p-nitrophenol. Since p-nitrophenol can be detectedspectrophotometrically, the enzymatic activity of the norbornane typeester hydrolase is measured by the following p-nitrophenyl acetatemethod in which p-nitrophenyl acetate (pNPA) is used as a substrate.

p-Nitrophenyl acetate method: an enzyme solution is added to a 0.1Mphosphate buffer (pH 7.0) including 0.02% p-nitrophenyl acetate and 5 mMmagnesium chloride so as to achieve a final volume of 3 ml. The reactionis conducted at a temperature of 30° C. for 20 minutes. The absorbanceof p-nitrophenol released into the reaction mixture by the enzymaticreaction is measured at 400 nm. The amount of the enzyme producing 1μmole of p-nitrophenol for 1 minute is defined as 1 unit (U).

(4) Properties of the enzyme:

The enzymatic properties and protein chemical properties of thenorbornane type ester hydrolase of the present invention are as follows:

(a) Enzymatic action and substrate specificity:

Various kinds of the (±)-exo-norbornane type esters respectivelycomprising formyl, acetyl, propionyl, n-butyryl or isobutyryl as theacyl R in formula I were allowed to come in contact with an enzymesolution, respectively as a substrate. Each of the obtained products wasextracted with an equal volume of chloroform. The extract was analyzedby gas chromatography using a column for optical resolution (produced byJ & W Scientific). (-)-Norborneol was selectively produced in all theextracts. As is apparent from this result, the enzyme is a hydrolasethat enantio-selectively hydrolyzes the (±)-exo-norbornane type ester toproduce an optically active norborneol, i.e., (-)-norborneol. In detail,referring to FIG. 1, (+)-(1S, 2S, 4R)-exo-norbornyl acetate ishydrolysed to the corresponding (-)-norborneol by the use of the presentenzyme. The obtained (-)-norborneol can be converted to (+)-d-norcamphorby oxidation. The remaining (-)-(1R, 2R, 4S)-exo-norbornyl acetate canbe hydrolysed to the corresponding (+)-norborneol by the use of analkali. The obtained (+)-norborneol can be converted to (-)-d-norcamphorby oxidation.

(b) Optimal pH and stable pH range:

The enzymatic activity measurement described in item 3 was performed atvarious pH values by using p-nitrophenyl acetate as a substrate. As aresult, the optimal pH for the reaction was found to be approximately 8.

The enzyme was allowed to stand for 1 hour at a temperature of 30° C. atvarious pH values. Then, the activity of the enzyme at each pH value wasmeasured by the method described in item 3. The results revealed thatthe stable pH range of the enzyme is approximately 6 to 8.

(c) Optimal temperature and thermal stability:

The enzymatic activity of the present enzyme was measured by the methoddescribed in item 3 at various temperatures. The results revealed thatthe optimal temperature was approximately 50° C.

The enzyme was allowed to stand in a phosphate buffer for 2 hours at pH7.0 at various temperatures. Then, the activity of the enzyme wasmeasured using the method described in item 3. The results revealed thatthe present enzyme was stable at a temperature up to 40° C. under theabove-mentioned conditions.

(d) Effect of inhibitors:

The present enzyme was allowed to stand in phosphate buffersrespectively including various inhibitors (5 mM) at pH 7.0 for 2 hours.Then, the activity of the enzyme was measured by the method described initem 3. The results revealed that the present enzyme was completelyinhibited by phenylmethylsulfonylfluoride (PMSF).

(e) Molecular weight:

The molecular weight of the present enzyme was measured by SDS-PAGE (SDSpolyacrylamide gel electrophoresis) using a gradient gel (produced byDaiichi Pure Chemicals Co., Ltd.) and a molecular weight marker(produced by Bio-Rad Lab.). The molecular weight was calculated to be 43kD.

(f) Amino acid composition:

The present enzyme was isolated by SDS-PAGE, and transferred onto a PVDFmembrane (produced by Milipore) by using a semidry blotting device(produced by Milipore). The resultant membrane was stained withCoomassie Brilliant Blue, and a visual portion corresponding to thepresent enzyme was cut out with a razor. The extract of the portion wassubjected to an amino acid analysis by HPLC using a picotag column(produced by Water Co., Ltd.). The results are shown in Table 1 below.Table 1 also shows the amino acid composition calculated from the aminoacid sequence determined based upon the DNA sequence of the presentenzyme described in detail below.

                  TABLE 1                                                         ______________________________________                                                Mole of Amino                                                         Amino   acid/mole of                                                          acid    protein (%)     Found   Calculated                                    ______________________________________                                        ASX     10.2            39.1    39                                            GLX     10.2            39.2    38                                            SER     4.4             17.1    22                                            GLY     8.2             31.4    33                                            HIS     2.0              7.7    12                                            ARG     2.8             10.8    12                                            THR     4.9             18.8    26                                            ALA     13.2            50.6    53                                            PRO     4.2             16.1    16                                            TYR     1.4              5.3     3                                            VAL     9.7             37.1    36                                            MET     2.2              8.4     8                                            CYS     0.0              0.0     1                                            ILE     5.5             21.0    17                                            LEU     13.3            50.9    41                                            PHE     3.6             13.8    11                                            TRP                      0.0     3                                            LYS     4.4             16.7    17                                            Total   100.0           384.0   388                                           ______________________________________                                    

(5) Determination of the DNA sequence encoding the norbornane type esterhydrolase:

The determination of the DNA sequence of a DNA fragment including DNAencoding the norbornane type ester hydrolase of the present invention isexemplified as follows: The sequence of the DNA fragment can bedetermined by analyzing the genomic DNA of Acetobacter pasteurianus.

(A) Cloning by the shotgun method:

Cells of Acetobacter pasteurianus are collected and treated with SDS.The resulting lysate is subjected to cesium chloride - ethidium bromideequilibrium density-gradient centrifugation to obtain chromosome DNA.The chromosome DNA is cleaved with an appropriate restriction enzyme.Each fragment of the cleaved DNA is inserted into a vector DNA that hasbeen previously cleaved with the same kind of restriction enzyme, orinserted, through a linker, into a vector that has been previouslycleaved with an appropriate restriction enzyme. Thus, recombinantplasmids are produced. Such a vector can be, for example, pBR322(produced by Takara Shuzo Co., Ltd.). The plasmids produced in thismanner are then introduced into host cells. A preferable host is E.coli, particularly, E. coli JM109 ATCC 53323. The recombinant plasmidsare introduced into host cells, for example, in accordance with themethod described by Hanahan, et al. (J. Mol. Biol., 166, 557-580(1983)).

(B) Selection of a clone including the gene of the norbornane type esterhydrolase:

A clone including the gene of the norbornane type ester hydrolase in theobtained transformant is screened as follows. The transformant issubject to a plate culture. The colonies grown thereon are transferredonto a filter paper. The filter paper is soaked with an esterasedetection solution which is a 50 mM phosphate buffer (pH 7.0) including0.1% β-naphthyl acetate and 0.02% Fast Blue BB. As a result, the colorof the colony in which esterase is produced is purplish red. Thus, acolony whose color has changed into purplish red is selected.

Such a colony is cultured, and allowed to react with 1%(±)-exo-norbornyl acetate. The product is analyzed by optical resolutiongas chromatography using the method described in item 4a. In thismanner, it is determined that the clone that has produced (-)-norborneolis a clone including the gene of the norbornane type ester hydrolase.

(C) Determination of the base sequence of an insert:

The base sequence of an insert of a recombinant plasmid is determined,for example, as follows: The insert is cleaved at a restriction enzymesite therein, and the obtained DNA fragments are respectively clonedwith appropriate vectors for sequence determination. The base sequenceof each of the cloned fragments is determined by the Sanger method(Sanger et al., Proc. Natl. Acad. Sci. U.S.A., 74, 5463-5467 (1977)).Thus, the entire base sequence of the insert can be determined.

(6) Construction of an expression vector including the gene of thenorbornane type ester hydrolase:

The gene of the norbornane type ester hydrolase of the present inventionis inserted into an appropriate vector such as pUC119 and pBR322(produced by Takara Shuzo Co., Ltd.) to serve as an expression vectorfor expressing the norbornane type ester hydrolase.

(7) Formation of a transformant and production of the norbornane typeester hydrolase:

The above-mentioned expression vector is introduced into a host cellsuch as E. coli, yeast and an animal cell to form a transformant. Byculturing the transformant, the norbornane type ester hydrolase of thepresent invention can be produced. The hydrolase is obtained as follows.

The transformant is cultured, and the culture is subjected tocentrifugation to collect the cells of the bacteria therein. Thecollected cells are washed with a buffer, and suspended in, for example,an equal volume of a 0.1M phosphate buffer (pH 7.0) to obtain a cellsuspension. The cells in the suspension are disrupted mechanically orultrasonically. The supernatant of the resultant suspension is obtainedby centrifugation as a crude enzyme solution.

The norbornane type ester hydrolase of the present invention can bepurified by various methods. An example of the methods includes thefollowing.

Ammonium sulfate is added to the crude enzyme solution so as to achieve30% saturation. The precipitate is removed from the mixture, andammonium sulfate is further added thereto so as to achieve 70%saturation. The precipitate in the mixture is collected. The dialyzedsolution of the collected precipitate is subjected to ion exchangechromatography using a DEAE Sepharose fast flow column and successivelygel filtration using a Sephacryl S-200 column to collect an activefraction. The fraction is subjected to polyacrylamide gelelectrophoresis by using a device produced by Marysol Industries. Theactivity of the resultant fraction is analyzed by the method describedin item 5B, and the active portion is cut out. The portion is crushedand eluted into a buffer to obtain an enzyme of high purity.

Any of the culture broth, the cell suspension, the crude enzyme and thepurified enzyme obtained in the above procedures can be used in theenzymatic reaction.

(8) Optically selective hydrolysis of the (±)-exo-norbornane type ester:

The (±)-exo-norbornane type ester is optically selectively hydrolyzed byusing the culture broth, the cell suspension, the crude enzyme or thepurified enzyme obtained in item 7. For example, the reaction isconducted in a buffer by mixing the (±)-exo-norbornane type ester withthe enzyme in one of the above forms at a temperature of 30° C. for 10minutes. Then, chloroform is added to the reaction mixture, and thesubstrate and product are extracted. The extract is analyzed by gaschromatography using, for example, a column for optical resolution(produced by J & W Scientific). Thus, it is confirmed that the opticallyactive norborneol is produced from the enzyme in any of the above forms.

EXAMPLES

The present invention will now be described in more detail by way ofexamples.

Example 1 Isolation and Purification of a Norbornane Type EsterHydrolase

A norbornane type ester hydrolase of the present invention was producedfrom Acetobacter pasteurianus ATCC 12873, and was isolated and purifiedas follows. Seventy liters of a medium (pH 7.0) containing 0.5% glucose,1.0% peptone, 1.0% yeast extract, 0.5% glycerin and 1.0% calciumcarbonate was charged in a 100 liter jar fermentor, and sterilized. Aseed culture of Acetobacter pasteurianus ATCC 12873, which had beenpreviously cultured in a medium (pH 7.0) containing 0.5% glucose, 1.0%peptone, 1.0% yeast extract and 0.5% glycerin, was inoculated thereto soas to make the volume of seed culture up to 4% of the entire medium. Thecultivation was conducted at a temperature of 28° C. for 48 hours, andthe culture, i.e., cell suspension, was centrifuged to collect thebacteria cells therein. The collected bacteria cells were washed twicewith a 0.1M phosphate buffer (pH 7.0) and suspended in 1.1 liter of a0.1M phosphate buffer. The suspension was treated three times with apressure type cell crusher (produced by Goulin Corp.; Manton-GoulinLaboratory homogenizer) to crush the cells therein. The resultant wascentrifuged and the supernatant was obtained.

Ammonium sulfate was added to the supernatant so as to achieve 30%saturation, and the mixture was stirred on ice for 1 hour andcentrifuged to obtain the supernatant. Ammonium sulfate was added tothis supernatant so as to achieve 70% saturation, and the mixture wasstirred on ice for 1 hour and centrifuged to collect the precipitate.The precipitate was dissolved in 320 ml of a 0.1M phosphate buffer (pH7.0), and the resultant solution was dialyzed against a 0.01M phosphatebuffer (pH 7.0) at 8° C. overnight.

To the dialysate, 1 liter of distilled water and 250 g of DEAE Sepharosefast flow (produced by Pharmacia) were successively added, and themixture was stirred at room temperature for 1 hour. The resultingSepharose resin was washed with 2 liters of a 0.01M phosphate buffer (pH7.0), and was packed into a column to elute an active fraction with a0.01M phosphate buffer (pH 7.0) containing 0.3M sodium chloride. Theeluted fraction was concentrated by using an ultrafiltration device(produced by Tosoh Corp.). The concentrate was loaded onto a column thathad been previously charged with Sephacryl S-200 (produced byPharmacia), equilibrated with a 50 mM Tris-HCl buffer, thereby elutingthe concentrate with 50 mM tris-HCl buffer. The eluted fraction wascollected and concentrated by the ultrafiltration.

The resulting concentrated solution was subjected to 10% polyacrylamidegel electrophoresis using a device produced by Marysol Industries. Afterthe electrophoresis, the obtained gel was subjected to the esteraseactivity staining method of Higerd and Spizizen (J. Bacteriol. 114,1184-1192 (1973)) as follows. The gel was dipped in 20 ml of a 0.1Mphosphate buffer (pH 7.0) including 10 mg β-naphthyl acetate and 10 mgof Fast Blue RR to detect an active portion which changed its color topurplish red. The purplish red portion was cut out from the gel, finelycrushed, and immersed in 100 ml of a 0.1M tris-HCl buffer at 4° C.overnight to elute an active protein. The procedure was repeated 10times to obtain 1 liter of the eluted solution. The eluted solution wasconcentrated by ultrafiltration to achieve a final volume of 10 ml.

Example 2 Measurement of the Activity of the Norbornane Type EsterHydrolase and Its Properties

The norbornane type ester hydrolase reacts with p-nitrophenyl acetate aswell as with exo-norbornane type ester as a substrate. The reaction withp-nitrophenyl acetate produces a colored material, p-nitrophenol. Sincep-nitrophenol can be detected spectrophotometrically, the enzymaticactivity of the norbornane type ester hydrolase was measured by the pNPAmethod in which p-nitrophenyl acetate was used as a substrate.Specifically, the reaction was conducted at 30° C. in 3 ml of a reactionmixture comprising 0.02% p-nitrophenyl acetate, a 0.1M phosphate buffer(pH 7.0) containing 5 mM magnesium chloride, and the enzyme solutionobtained in Example 1. Free p-nitrophenol was quantified by measuringthe absorbance at 400 nm by a spectrophotometer. The amount of enzymenecessary to produce 1 μmole of p-nitrophenol for 1 minute was definedas 1 unit (U). As a result, the activity of the enzyme purified inExample 1 was found to be 38.7 U/ml. Further, the amount of proteinmeasured by a protein assay kit (produced by Bio-Rad Lab.) was 0.25mg/ml.

The purified enzyme was analyzed in accordance with the method ofLaemmli (Nature, 227, 680-685 (1970)) by SDS-PAGE using a gradient gel(produced by Daiichi Pure Chemicals Co., Ltd. ) and a molecular weightmarker (produced by Bio-Rad Lab. ). As a result, the main band was foundat a position corresponding to a molecular weight of 43 kD. Further, theenzyme was analyzed by HPLC using HPLC molecular weight markers(produced by Oriental Yeast Co., Ltd.) and a molecular weight measuringcolumn (produced by Waters Co., Ltd.; PROTEINPAK-300). In this analysis,the active portion was eluted at a position also corresponding to amolecular weight of 43 kD. Therefore, it was indicated that thenorbornane type ester hydrolase existed as a monomer.

The principal properties of the enzyme were found as follows:

(1) Appropriate reaction temperature and optimal pH:

The above-mentioned enzymatic activity measurement was performed byusing p-nitrophenyl acetate as a substrate at various pH values. As aresult, the optimal pH was found to be approximately 8. Further, theenzymatic activity measurement was also performed at varioustemperatures to find the appropriate reaction temperature to beapproximately 50° C.

(2) Heat stability and a stable pH range:

One ml of the enzyme solution including a 0.1M phosphate buffer (pH 7.0)was allowed to stand for 2 hours at various temperatures, and thensubjected to the above-mentioned activity measurement. As a result, theenzyme was found to be stable at a temperature up to 40° C. under theabove-mentioned conditions. Furthermore, 1 ml of the enzyme solutionincluding a 0.1M buffer at various pH values was allowed to stand at 30°C. for 1 hour, and then subjected to the activity measurement. As aresult, the enzyme was found to be stable in a pH range of 6 to 8.

(3) Effect of inhibitors:

The above-mentioned activity measurement was performed with regard to0.5 ml of the enzyme solution of 0.1M phosphate buffer (pH 7.0)containing 5 mM of one of the following inhibitors:ethylenediaminetetraacetic acid (EDTA), phenylmethylsulfonylfluoride(PMSF), N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK),N-tosyl-L-lysyl chloromethyl ketone (TLCK), diisopropyl fluorophosphate(DFP), N-ethylmaleimide and iodoacetic acid. The enzyme solutions,including the respective inhibitors, were allowed to stand at 30° C. for2 hours. As a result, the present enzyme was found to be completelyinhibited by PMSF.

(4) Stability in solvents:

The activity measurement was performed with regard to 0.2 ml of theenzyme solution of 0.1M phosphate buffer (pH 7.0) containing one of thefollowing solvents at a concentration of 30%: ethanol, methanol,acetonitrile, acetone, dimethyl sulfoxide (DMSO), N,N-dimethylformamide, 1,4-dioxane, ethyl acetate and trichloromethane.The enzyme solutions including the respective solvents were allowed tostand at 30° C. for 1 hour. As a result, the present enzyme exhibited80% or more activity in methanol, DMSO or ethyl acetate at aconcentration of 30%.

(5) Optically selective hydrolysis of the norbornane type ester:

The purified enzyme obtained in Example 1 was allowed to come in contactwith various kinds of 1% (±)-exo-norbornane type esters respectivelyincluding formyl, acetyl, propionyl, n-butyryl and isobutyryl as theacyl R in formula I. The reaction was conducted at 30° C. for 30minutes. The resultant was extracted with an equal volume of chloroform.The extract was analyzed by gas chromatography using a column foroptical resolution (produced by J & W Scientific). As is apparent fromthe results shown in Table 2, (-)-norborneol was enantio-selectivelyproduced from any of the esters.

                  TABLE 2                                                         ______________________________________                                        Acyl R in the norbornane                                                                      Produced norborneol (mg/ml)                                   type ester Formula I                                                                          (-)-isomer   (+)-isomer                                       ______________________________________                                        Formyl          1.49         0.15                                             Acetyl          2.48         0.19                                             Propionyl       3.39         0.45                                             n-butyryl       2.30         0.29                                             isobutyryl      2.76         0.69                                             ______________________________________                                    

Example 3 Cloning of the Gene of the Norbornane Type Ester Hydrolasefrom Acetobacter pasteurianus

(1) Cloning by the shotgun method:

Cells of Acetobacter pasteurianus in a culture medium were collected,and treated with 1% SDS to obtain a lysate. The lysate was subjected tocesium chloride - ethidium bromide equilibrium density-gradientcentrifugation, thereby obtaining purified chromosomal DNA.Approximately 100 units of a restriction enzyme BamHI was added to 10 μgof the DNA, and the reaction was conducted at 37° C. for 120 minutes.The resultant mixture was incubated at 70° C. for 5 minutes toinactivate the restriction enzyme, and ethanol precipitation wasperformed to collect the DNA. The obtained DNA fragments, cleaved withthe restriction enzyme BamHI, were dissolved in 10 μl of TE buffer of 10mM Tris-HCl (pH 8.0) including 1 mM EDTA.

A vector pBR322 (produced by Takara Shuzo Co., Ltd. ) was used as acloning vector. Approximately 50 units of the restriction enzyme BamHIwas added to 5 μg of the vector pBR322, and the reaction was conductedat a temperature of 37° C. for 2 hours. The reaction mixture wasprecipitated with ethanol to collect the DNA. The collected DNA wasdissolved in a buffer (pH 8.0) including 50 mM of Tris-HCl and 1 mMmagnesium chloride. Approximately 20 units of bacterial alkalinephosphatase (BAP) was added thereto, and the reaction was conducted at37° C. for 1 hour. The resultant reaction mixture was extracted with anequal volume of a phenol solution. Ethanol was added to the obtainedaqueous layer to precipitate the DNA. The collected DNA was dissolved in10 μl of TE buffer.

Five μl of the obtained solution including the fragments of thechromosome of Acetobacter pasteurianus, cleaved with the restrictionenzyme BamHI, was mixed with 1 μl of a solution including the vectorpBR322 cleaved and dephosphorylated with the restriction enzyme BamHIand BAP. Ligation was achieved with a commercially available kit(produced by Takara Shuzo Co., Ltd.; DNA Ligation Kit) to produce arecombinant DNA. Using the recombinant DNA, E. coli JM109 ATCC 53323 wastransformed in accordance with the method of Hanahan et al. (J. Mol.Biol., 166, 557-580 (1983)). The transformant was cultured on an L agarmedium (pH 7.3) (i.e., a medium including 1% bacto tryptone, 0.5% yeastextract, 0.5% sodium chloride and 1.8% agar) including 50 μg/ml ofampicillin, whereby colonies of the transformant were formed.

(2) Selection of the clone:

The activity staining method of Example 1 was used to select a colonyhaving an esterase activity among the colonies formed in item 1 above.Specifically, the colonies formed on the L agar medium were replicatedonto a filter paper. The filter paper was soaked with an esterasedetection solution which is a 50 mM phosphate buffer (pH 7.0 ) including0.1% β-naphthyl acetate and 0.02% Fast Blue BB to obtain a purplish redcolony exhibiting esterase activity. Through this procedure, 17 purplishred colonies were detected and isolated from approximately 7,800colonies.

Such an positive colony was cultured overnight by using 5 ml of an Lbroth including 50 μg/ml of ampicillin to react with 1% exo-norbornylacetate. As a result, norborneol was produced. In this manner, thestrain forming the positive colony was confirmed to be a clone includingthe gene of the norbornane type ester hydrolase.

From the cell of the strain producing the hydrolase, plasmids wereisolated by the method of Birnboim et al. (Nucleic Acids Res., 7,1513-1523 (1979)). The plasmids were cleaved with the restrictionenzymes BamHI, EcoRI, HindIII and PstI. The resultant recombinantplasmids were analyzed by agarose gel electrophoresis. As a result, itwas found that a fragment of 4.5 kb was inserted in the same directionin all of the plasmids. One of the plasmids was designated as pBNA21.

(3) Preparation of the restriction map of the plasmid and minimizationof the plasmid:

The recombinant plasmid pBNA21 was cleaved with various restrictionenzymes to construct a restriction map. The resulting restriction map isshown in FIG. 2. The abbreviations used in this figure indicate sites tobe cleaved with the following restriction enzymes:

E: EcoRI

H: HindIll

EV: EcoRV

B: BamHI

P: PstI

Nc: NcoI

Af: Af1II

S: Sa1I

Further, in order to determine the location of the gene encoding thenorbornane type ester hydrolase, deletions and frameshift mutations ofan exogeneous DNA fragment of 4.5 kb were prepared by using therestriction enzymes BamHI, HindIII, Af1II, NcoI and Sa1I. The resultantDNA fragments were analyzed by the esterase activity staining method ofitem 2 to find whether or not they had the enzymatic activity of thenorbornane type ester hydrolase. As a result, it was found that only theplasmid in which an Af1II-Sa1I fragment of 2.4 kb was deleted did notexhibit the activity. This result revealed that the gene of thenorbornane type ester hydrolase of the present invention was located ina region of 2.1 kb between Af1II and BamHI that corresponds to theAf1II-Sa1I fragment of 2.4 kb excluding 0.3 kb between BamHI and Sa1Iderived from the vector.

Then, the plasmid pBNA21 was cleaved with the restriction enzyme Af1II,blunt ended with T4 DNA polymerase, and then cleaved with therestriction enzyme BamHI. The resultant reaction mixture was subjectedto agarose gel electrophoresis to obtain a DNA fragment of 2.1 kb byusing a commercially available kit (produced by BIO101; GENECLEAN).

A vector pUC119 (produced by Takara Shuzo Co., Ltd.) was cleaved atBamHI and HincII cleavage sites existing at a multicloning site of thelacZ gene in the vector. The DNA fragment of 2.1 kb was ligated with theresultant vector fragment so that the fragment is aligned in the samedirection as that of the lac promoter of the vector pUC119. Since theblunt ended Af1II cleavage site was ligated with the HincII cleavagesite, the Af1II cleavage site was regenerated. E. coli JM109 wastransformed by using the obtained recombinant plasmid, and the resultingtransformant was cultured to form colonies having the activity of thenorbornane type ester hydrolase. The recombinant plasmid produced inthis manner was designated as pUNA221. The restriction map of pUNA221 isshown in FIG. 3.

Example 4 Determination of the Base Sequence of the Gene of theNorbornane Type Ester Hydrolase, and Identification of the StructuralGene of the Norbornane Type Ester Hydrolase

The DNA base sequence of the Af1II-BamHI fragment of 2.1 kb in therecombinant plasmid pUNA221 produced in Example 3 was determined by themethod of Sanger et al. (Proc. Natl. Acad. Sci. U.S.A., 74, 5463-5467(1977)). As a result, an open reading frame (ORF) encoding a proteincomprising 388 amino acids including methionine corresponding to thestarting codon ATG was found.

The Af1II-BamHI fragment of 2.1 kb was then cleaved with a restrictionenzyme HinfI to obtain a fragment of 1.5 kb including the ORF that wasregarded as encoding the norbornane type ester hydrolase. The obtainedfragment was inserted into a plasmid pUC119 at the HincII cleavage sitein the same direction as that of the lac promoter, thereby producing aplasmid pUNA-Hf.

E. coli JM109 was transformed by using the plasmid pUNA-Hf. When theobtained transformant was analyzed by the activity staining methoddescribed in item 2 of Example 3, the color of the colony was changedinto purplish red to exhibit esterase activity. Then, the transformantwas mixed with a 1% (±)-exo-norbornyl acetate mixture and allowed tostand at 30° C. for 10 minutes. The resulting mixture was analyzed bythe method described in item 5 of Example 2. As a result, it was foundthat 1.68 mg/ml of (-)-exo-norborneol and 0.15 mg/ml of(+)-exo-norborneol were produced. Thus, the optical selectivity wasconfirmed to be as high as that of the enzyme derived from Acetobacterpasteurianus obtained in Example 1.

Example 5 Expression of the Norbornane Type Ester Hydrolase in E. coli

(1) Expression of the norbornane type ester hydrolase

The plasmid pUNA-Hf obtained in Example 4 was introduced into E. coliJM103 ATCC 39403 to produce a transformant JM103/pUNA-Hf.

The transformant JM103/pUNA-Hf, which was previously cultured in an Lbroth including 50 μg/ml of ampicillin, was inoculated into 50 ml of thesame medium in a 500 ml flask so that the inoculum size of thetransformant was 1% of the entire medium, and was cultured at 37° C.with shaking. When OD₆₆₀ was 0.2, 1 mM isopropyl-β-D-galactopyranoside(IPTG) was added thereto. The cultivation was continued, and 6 hoursafter the addition of IPTG, the culture broth was obtained. The culturebroth was frozen overnight, and melted to be used as a specimen for anactivity measurement. The activity measured by the pNPA method was 68units per 1 ml of the culture broth. As a control, a strain includingthe plasmid pUC119 alone was cultured in the same manner as above. Theactivity of the control was 0.2 unit per 1 ml of the culture solution.

(2) Optically selective deacetylation of exo-norbornyl acetate:

(±)-Exo-norbornyl acetate used as a substrate was optically selectivelydeacetylated by E. coli JM103 including the plasmid pUNA-Hf as follows.The bacterial cells were collected from the cultured broth of E. coliJM103/pUNA-Hf as mentioned above by centrifugation, and suspended in anequal volume of a 0.1M phosphate buffer (pH 7.0). Then, 1%(±)-exo-norbornyl acetate was added to 1.5 ml of the obtainedsuspension, and the reaction was conducted at 30° C. for 15 minutes. Theresultant reaction mixture was analyzed by the method described in item5 of Example 2. As a result, it was found that 1.91 mg/ml of(-)-exo-norborneol and 0.15 mg/ml of (+)-exo-norborneol were produced.

Examples of the usage of the optically active norborneol produced fromthe norbornane type ester hydrolase of the present invention will now bedescribed as Reference Examples.

Reference Example 1

By the use of a norbornane type ester hydrolase obtained in Example 1,(-)-(1S, 2S, 4R)-exo-norborneol was produced according to the method ofitem 5 in Example 2. Then, 2.8 g (0.025 mole) of the obtained (-)-(1S,2S, 4R)-exo-norborneol was dissolved in 56 ml of methylene chloride, and8.1 g (1.5 mole) of pyridinium chlorochromate (PCC) and 1 g of molecularsieve 4A were added thereto. The reaction was conducted at a temperatureof 25° C. to 30° C. for 1 hour. Then, the reaction mixture was dilutedwith 56 ml of toluene, and the resultant was allowed to pass through acolumn of 28 g silica gel to remove insoluble materials. The eluate wasconcentrated and dried to give crude (+)-norcamphor as a white crystalline powder. The obtained (+)-norcamphor was dissolved in 45 ml oftetrahydrofuran, and the resultant solution was added dropwise to a 30ml tetrahydrofuran solution including 1.1 mole of Lithium diisopropylamide (LDA) at a temperature of -10° C. to -15° C. The reaction wasconducted at the same temperature for 20 minutes, and 3.3 g (1.1 molarratio) of allyl bromide was added dropwise thereto at 0° C. or lower.Then, the reaction was conducted at room temperature for 3 hours. Thereaction mixture was poured into 70 ml of ice water, and the resultantmixture was made acidic with a diluted hydrochloric acid solution. Theacidic solution was extracted with 50 ml of toluene twice. The combinedtoluene layer was washed with water, and the solvent was removed invacuo to give crude (+)-exo-3-(2-propenyl)-bicyclo[2.2.1]heptan-2-one asan oily residue. The residue was distilled in vacuo to obtain a fractionhaving a boiling point of 92° C.-103° C. at 10-12 mmHg. Yield: 3.07 g(82%). Chemical purity (GC): 98.6%. Endo isomer: 0.4%. Optical purity(HPLC): 90% ee. Specific rotation [α]²⁵ D+84.6° (C=1.0, CHCl₃).

Reference Example 2

The similar procedure to Example 5 was carried out on a large scale, anda reaction mixture including a major amount of (-)-(1S, 2S,4R)-exo-norborneol and a minor amount of (+)-(1R, 2R, 4S)-exo-norborneolwas obtained. The similar procedure to that described in ReferenceExample 1 was repeated with the use of this mixture. Thus, a crude(+)-exo-3-(2-propenyl)-bicyclo[2.2.1]heptan-2-one was obtained. Thecrude product was distilled in vacuo to obtain a fraction having aboiling point of 74° C.-86° C. at 3-4 mmHg which is a purified(+)-exo-3-(2-propenyl)-bicyclo[2.2.1]heptan-2-one. Yield: 1.38 g (78%).Chemical purity (GC): 98.8%. Endo isomer: 0.9%. Optical purity (HPCL):98% ee. Specific rotation [α]²⁵ D+89° (C=1.286, CHCl₃). IR (Film) 3060,1740, 1640, 1460, 1440, 1310, 1090 cm⁻¹. ¹ HNMR (CDCl₃) δ 1.30-2.00 (m,8H), 2.5-2.6 (m, 3H) δ 4.90-5.20 (m, 2H), 5.7-5.9 (m, 1H).

The compounds obtained in the Reference Examples can be used to producea TXA₂ receptor antagonist that is useful as a medicament by the methoddescribed for example, in J. Med. Chem. 31(9), 1847-1854 (1988).

As described above, the present invention provides a norbornane typeester hydrolase that enantio-selectively hydrolyzes a (±)-exo-norbornanetype ester to produce optically active norborneol; a structural gene ofthe norbornane type ester hydrolase derived from Acetobacterpasteurianus; an expression vector including the structural gene; atransformant including the expression vector; and a production methodfor the norbornane type ester hydrolase using the transformant. Theoptically active norborneol produced by reacting the norbornane typeester hydrolase of the present invention with a (±)-exo-norbornane typeester can be synthesyzed into(±)-exo-3-(2-propenyl)-bicyclo[2.2.1]heptan-2-one, from which amedically useful TXA₂ receptor antagonist can be produced.

Various other modifications will be apparent to and can be readily madeby those skilled in the art without departing from the scope and spiritof this invention. Accordingly, it is not intended that the scope of theclaims appended hereto be limited to the description as set forthherein, but rather that the claims be broadly construed.

The following specific sequence information and descriptions areprovided in order to comply with the formal requirements of thesubmission of sequence data to the United States Patent and TrademarkOffice and are not intended to limit the scope of what the inventorsregard as their invention. Variations in sequences which will becomeapparent to those skilled in the art upon review of this disclosure andwhich are encompassed by the attached claims are intended to be withinthe scope of the present invention. Further, it should be noted thatefforts have been made to insure accuracy with respect to the specificsequences and characteristic description information describing suchsequences, but some experimental error and/or deviation should beaccounted for.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1167 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Acetobacter pasteurianus                                       (B) STRAIN: ATCC 12873                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1164                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATGTCCAACACCATTACAGCACTCACCATGCCCAAGTTCGGTTTGGCC48                            MetSerAsnThrIle ThrAlaLeuThrMetProLysPheGlyLeuAla                             151015                                                                        ATGACAGAAGGCAAGCTGGCATCATGGACAGTATCTGTTGGCCAGAGC96                            MetThrGluGly LysLeuAlaSerTrpThrValSerValGlyGlnSer                             202530                                                                        GTGCAGCAGGGTGATGAACTGGCGGATATTGAAACCACCAAAATTACC144                           ValGlnGlnGly AspGluLeuAlaAspIleGluThrThrLysIleThr                             354045                                                                        AGCAGTTATGAAAGCCCCGCCGCAGGTGTGCTGCGCAAACAGGTGGCC192                           SerSerTyrGluSer ProAlaAlaGlyValLeuArgLysGlnValAla                             505560                                                                        GAGGCGGGGGAAACCCTGCCCGTGGGCGCACTGATTGGTGTTTTGGCA240                           GluAlaGlyGluThrLeuPro ValGlyAlaLeuIleGlyValLeuAla                             65707580                                                                      GATGCCGAAACGCCAGATGTGGATATTGAAGCCTTTATCAAAAACTTT288                           AspAlaGluThrPro AspValAspIleGluAlaPheIleLysAsnPhe                             859095                                                                        CATGCAGAAAACCCACAGGATGCGGCTGCAACAGAAGATGCCTCTGCC336                           HisAlaGluAsn ProGlnAspAlaAlaAlaThrGluAspAlaSerAla                             100105110                                                                     GGGGAACCCAAGCAGGTTACGGTAGGCGAACACACGCTAAATGTGCGT384                           GlyGluProLys GlnValThrValGlyGluHisThrLeuAsnValArg                             115120125                                                                     GATGTTGGCACGCAGGAGGGCACGCCCATTGTGCTGGTGCACGGTTTT432                           AspValGlyThrGln GluGlyThrProIleValLeuValHisGlyPhe                             130135140                                                                     GGCGGAGATATCAGCAACTGGCTGCTCACACAGGATGCCTTGGCCGCA480                           GlyGlyAspIleSerAsnTrp LeuLeuThrGlnAspAlaLeuAlaAla                             145150155160                                                                  GAAAGGCGCGTAATTGCGTTTGATCTGCCGGGGCATGGGGCTTCCTCT528                           GluArgArgValIle AlaPheAspLeuProGlyHisGlyAlaSerSer                             165170175                                                                     AAAAACGTGGGCACAGGCACGCTGGCGTTTTTGGCCGGTGTGGTAAGC576                           LysAsnValGly ThrGlyThrLeuAlaPheLeuAlaGlyValValSer                             180185190                                                                     GAATTGCTGCAAACCCTTAAAATAGAAAAAGCCCATGTGGTGGGCCAT624                           GluLeuLeuGln ThrLeuLysIleGluLysAlaHisValValGlyHis                             195200205                                                                     TCTTTGGGGGGCGGCATTGCCCTGACCCTGCTGCGAGATCACCCTGAT672                           SerLeuGlyGlyGly IleAlaLeuThrLeuLeuArgAspHisProAsp                             210215220                                                                     CAGGTTGCCAGCCTGAACCTTTTGGCCCCAGCCGGGTTGGGTAAGGAT720                           GlnValAlaSerLeuAsnLeu LeuAlaProAlaGlyLeuGlyLysAsp                             225230235240                                                                  GTGAATGCAGATTTTATCAGCGCATTTGTGGATAGTGAAAGCAGCCGC768                           ValAsnAlaAspPhe IleSerAlaPheValAspSerGluSerSerArg                             245250255                                                                     GATATGAAGGCTGTTTTGCAAATGCTGGTGTATAACAAAGCCCTAGTG816                           AspMetLysAla ValLeuGlnMetLeuValTyrAsnLysAlaLeuVal                             260265270                                                                     GGCCGTAAGATGGTGGATGCCGTGCTGCGTGCACGTAGGCTAGATGGC864                           GlyArgLysMet ValAspAlaValLeuArgAlaArgArgLeuAspGly                             275280285                                                                     GCGCGGGATGCCCTGCACGTTATTGCTAAAGCGTGCTTCCCCAACGGG912                           AlaArgAspAlaLeu HisValIleAlaLysAlaCysPheProAsnGly                             290295300                                                                     CATCAGGCGGATGATCTGCACTCGGTGCTAGCTGGGGCGGAAACACCT960                           HisGlnAlaAspAspLeuHis SerValLeuAlaGlyAlaGluThrPro                             305310315320                                                                  ACCCAGATTTTCTGGGGCAAGGAAGATGAAATTCTTTCTGTCTCCAAC1008                          ThrGlnIlePheTrp GlyLysGluAspGluIleLeuSerValSerAsn                             325330335                                                                     GCCGCTGGCCTGCCAGATGTCATCCCCGTGACAGTGTATGAAGAAACA1056                          AlaAlaGlyLeu ProAspValIleProValThrValTyrGluGluThr                             340345350                                                                     GGCCATCTGCCGCAGCTTGAACATGCAACAGATGTGAACAAAGCCATT1104                          GlyHisLeuPro GlnLeuGluHisAlaThrAspValAsnLysAlaIle                             355360365                                                                     GCCCTGTTTGTAAAAGACCCCGAAGCCGCGCTGAGCATGGCCCGGATG1152                          AlaLeuPheValLys AspProGluAlaAlaLeuSerMetAlaArgMet                             370375380                                                                     GACGCGACAGCCTAA1167                                                           AspAlaThrAla                                                                  385                                                                           (2 ) INFORMATION FOR SEQ ID NO:2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 388 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetSerAsnThrIleThrAlaLeuThrMetProLysPheGlyLeuAla                              15 1015                                                                       MetThrGluGlyLysLeuAlaSerTrpThrValSerValGlyGlnSer                              202530                                                                        ValGlnGlnGlyAspGluLeuAlaA spIleGluThrThrLysIleThr                             354045                                                                        SerSerTyrGluSerProAlaAlaGlyValLeuArgLysGlnValAla                              50556 0                                                                       GluAlaGlyGluThrLeuProValGlyAlaLeuIleGlyValLeuAla                              65707580                                                                      AspAlaGluThrProAspValAspIleGluAlaPheIleLysAsnPh e                             859095                                                                        HisAlaGluAsnProGlnAspAlaAlaAlaThrGluAspAlaSerAla                              100105110                                                                     GlyGlu ProLysGlnValThrValGlyGluHisThrLeuAsnValArg                             115120125                                                                     AspValGlyThrGlnGluGlyThrProIleValLeuValHisGlyPhe                              130 135140                                                                    GlyGlyAspIleSerAsnTrpLeuLeuThrGlnAspAlaLeuAlaAla                              145150155160                                                                  GluArgArgValIleAlaPheAspLeuP roGlyHisGlyAlaSerSer                             165170175                                                                     LysAsnValGlyThrGlyThrLeuAlaPheLeuAlaGlyValValSer                              180185 190                                                                    GluLeuLeuGlnThrLeuLysIleGluLysAlaHisValValGlyHis                              195200205                                                                     SerLeuGlyGlyGlyIleAlaLeuThrLeuLeuArgAspHisProAs p                             210215220                                                                     GlnValAlaSerLeuAsnLeuLeuAlaProAlaGlyLeuGlyLysAsp                              225230235240                                                                  ValAsnAla AspPheIleSerAlaPheValAspSerGluSerSerArg                             245250255                                                                     AspMetLysAlaValLeuGlnMetLeuValTyrAsnLysAlaLeuVal                              260 265270                                                                    GlyArgLysMetValAspAlaValLeuArgAlaArgArgLeuAspGly                              275280285                                                                     AlaArgAspAlaLeuHisValIleAlaL ysAlaCysPheProAsnGly                             290295300                                                                     HisGlnAlaAspAspLeuHisSerValLeuAlaGlyAlaGluThrPro                              305310315 320                                                                 ThrGlnIlePheTrpGlyLysGluAspGluIleLeuSerValSerAsn                              325330335                                                                     AlaAlaGlyLeuProAspValIleProValThrValTyrGluGl uThr                             340345350                                                                     GlyHisLeuProGlnLeuGluHisAlaThrAspValAsnLysAlaIle                              355360365                                                                     AlaLeuPhe ValLysAspProGluAlaAlaLeuSerMetAlaArgMet                             370375380                                                                     AspAlaThrAla                                                                  385                                                                       

What is claimed is:
 1. An isolated or purified norbornane type esterhydrolase derived from a bacterium of the species Acetobacterpasteurianus that enantioselectively hydrolyzes a (±)-exo-norbornanetype ester represented by Formula I: ##STR6## wherein R is acyl, and Aand B are hydrogens, respectively, or where A and B are absent,resulting in a carbon-carbon double bond between the carbons to which Aand B are attached in Formula I;the norbornane type ester hydrolasehaving an optimal pH of approximately 8 and a stable pH range ofapproximately 6 to
 8. 2. An isolated or purified norbornane type esterhydrolase derived from a bacterium of the species Acetobacterpasteurianus that enantioselectively deacetylates a (±)-exo-norbornylacetate represented by Formula II: ##STR7## the norbornane type esterhydrolase having an optimal pH of approximately 8 and a stable pH rangeof approximately 6 to
 8. 3. An isolated or purified norbornane typeester hydrolase according to claim 1, wherein the Acetobacterpasteurianus is Acetobacter pasteurianus ATCC
 12873. 4. An isolated orpurified norbornane type ester hydrolase according to claim 2, whereinthe Acetobacter pasteurianus is Acetobacter pasteurianus ATCC
 12873. 5.An isolated or purified norbornane type ester hydrolase comprising anamino acid sequence from Met in the 1 position to Ala in the 388position of SEQ ID No. 1.